AAV-mediated delivery of DNA to cells of the nervous system

ABSTRACT

The invention relates to a method of delivering exogenous DNA to a target cell of the mammalian central nervous system using an adeno-associated virus (AAV)-derived vector. Also included in the invention are the AAV-derived vectors containing exogenous DNA which encodes a protein or proteins which treat nervous system disease, and a method of treating such disease.

RELATED APPLICATION

This application is a continuation-in-part of application U.S. Ser. No.08/227,319 filed on Apr. 13, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the delivery of DNA to and theexpression of delivered genes in, cells of the nervous system.

2. Description of the Related Art

The first human gene therapy trial started in September 1990 andinvolved retrovirally-mediated transfer of the adenosine deaminase (ADA)gene into lymphocytes of patients with severe combined immunodeficiency(SCID). The favorable results of this trial stimulated further interestin gene therapy resulting in further 67 gene therapy clinical protocolsapproved by the NIH Recombinant DNA Advisory Committee (RAC) to date.Although the original promise of gene therapy was the development of acurative treatment for simple, single gene diseases, the vast majorityof gene therapy trials have been for complex genetic or acquireddiseases, such as infectious disease and cancer. A large number of theinitial clinical gene transfer studies were not gene therapy but rathergene marking studies. The first type of marking experiments used tumorinfiltrating lymphocytes which were transduced in vitro with retroviralvectors prior to infusion into patients with cancer. The second class ofgene marking studies involved the attempt to detect residual tumor cellsin marrow infused into patients following ablative chemotherapy.

Of the currently approved gene therapy trials, all trials prior to 1992used retroviral vectors and the diseases targeted included SCID,familial hypercholesteremia and cancer. More recently, gene therapytrials have commenced for AIDS and Hemophilia B, again using retroviralvectors. In addition, adenoviral vectors have recently been approved forcystic fibrosis. The vast majority of these protocols have enrolled veryfew patients at present and most of the trials are as yet unpublished.The available data however appears promising, for example the expressionof the LDL receptor in the liver following ex vivo transduction ofresected hepatocytes and its infusion into the portal vein in patientswith familial hypercholesteremia has resulted in a 20% drop in plasmacholesterol levels (Randall (1992) JAMA 269:837-838). It is likelytherefore that there will be an exponential growth in gene therapytrials and a large number of medical schools and teaching hospitals aresetting up gene therapy centers.

The ability to deliver genes to the nervous system, and to manipulatetheir expression, may make possible the treatment of numerousneurological disorders. Unfortunately, gene transfer into the centralnervous system (CNS) presents several problems including the relativeinaccessibility of the brain and the blood-brain-barrier, and thatneurons of the postnatal brain are post-mitotic. The standard approachfor somatic cell gene transfer, i.e., that of retroviral vectors, is notfeasible for the brain, as retrovirally mediated gene transfer requiresat least one cell division for integration and expression. A number ofnew vectors and non-viral methods have therefore been used for genetransfer in the CNS. Although the first studies of gene transfer in theCNS used an ex vivo approach, i.e., the transplantation ofretrovirally-transduced cells, more recently several groups have alsoused an in vivo approach. Investigators have used HSV-1 and adenoviralvectors as well as non-viral methods including cationic lipid mediatedtransfection (Wolff (1993) Curr. Opin. Biol. 3:743-748).

The ex vivo approach is illustrated by a recent study in whicholigodendrocytes were retrovirally infected and transplanted into asyngeneic rat model for demyelination (Groves et al (1993) Nature362:453-457). In addition to the use of brain cells as vehicles forforeign gene expression in the CNS, non-neuronal cells includingfibroblasts and primary muscle cells have also been used (Horrelou et al(1990) Neuron 5:393-402; Jiao et al (1993) Nature 362:450-453).

The in vivo approach was initially largely based on the use of theneurotropic Herpes Simplex Virus (HSV-1), however, HSV vectors presentseveral problems, including instability of expression and reversion towild-type (see below). A more recent development has been the use ofadenoviral vectors. Adenoviral vector studies have shown expression ofmarker genes into the rat brain persisting for two months althoughexpression fell off dramatically (Davidson et al (1993) Nature Genetics3:219-2223). In addition to viral vector approaches, other investigatorshave used direct injection of a cationic liposome:plasmid complexobtaining low level and transient expression of a marker gene (Ono et al(1990) Neurosci. Lett. 117:259-263).

There have been very few studies using “therapeutic” genes in the CNS.The majority of these have used the ex vivo approach with transductionof fibroblasts and muscle cells with the human tyrosine hydroxylase genein order to produce L-dopa-secreting cells for use in models ofParkinson's Disease (e.g., Horrelou et al (1990) Neuron 5:393-402; Jiaoet al (1993) Nature 362:450-453). Of the in vivo approaches, HSV vectorshave been used to express β-glucuronidase (Wolfe et al (1992) NatureGenetics 1:379-384), glucose transporter (Ho et al (1993) Proc. Natl.Acad. Sci. 90:6791-6795) and nerve growth factor (Federoff et al (1992)Proc. Natl. Acad. Sci. 89:1636-1640). An adenoviral vector has been usedto induce low level transient expression of human al-antitrypsin(Bajoccchi et al (1993) 3:229-234).

The only clinical studies of gene transfer in the brain followed areport by Culver et al (1992) Science 256:18550-18522) in which theyessentially cured rats which had been intracerebrally implanted withglioma cell lines. They used a retrovirus expressing the HSV-1 thymidinekinase (tk) gene and then subsequently treated with ganciclovir. In1993, a human protocol for glioblastoma multiforme was approved usingthe retroviral tk vector-ganciclovir protocol (Oldfield et al (1993)Human Gene Ther. 4:39-69).

Herpes viruses

The genome of the herpes simplex virus type-1 (HSV-1) is about 150 kb oflinear, double-stranded DNA, featuring about 70 genes. Many viral genesmay be deleted without the virus losing its ability to propagate. The“immediately early” (IE) genes are transcribed first. They encodetrans-acting factors which regulate expression of other viral genes. The“early” (E) gene products participate in replication of viral DNA. Thelate genes encode the structural components of the virion as well asproteins which turns on transcription of the IE and E genes or disrupthost cell protein translation.

After viral entry into the nucleus of a neuron, the viral DNA can entera state of latency, existing as circular episomal elements in thenucleus. While in the latent state, its transcriptional activity isreduced. If the virus does not enter latency, or if it is reactivated,the virus produces numerous infectious particles, which leads rapidly tothe death of the neuron. HSV-1 is efficiently transported betweensynaptically connected neurons, and hence can spread rapidly through thenervous system.

Two types of HSV vectors have been utilized for gene transfer into thenervous system. Recombinant HSV vectors involve the removal of animmediate-early gene within the HSV genome (ICP4, for example), andreplacement with the gene of interest. Although removal of this geneprevents replication and spread of the virus within cells which do notcomplement for the missing HSV protein, all of the other genes withinthe HSV genome are retained. Replication and spread of such viruses invivo is thereby limited, but expression of viral genes within infectedcells continues. Several of the viral expression products may bedirectly toxic to the recipient cell, and expression of viral geneswithin cells expressing MHC antigens can induce harmful immunereactions. In addition, nearly all adults harbor latent herpes simplexviruses within neurons, and the presence of recombinant HSV vectorscould result in recombinations which can produce an actively replicatingwild-type virus. Alternatively, expression of viral genes from therecombinant vector within a cell harboring a latent virus might promotereactivation of the virus. Finally, long-term expression from therecombinant HSV vector has not been reliably demonstrated. It is likelythat, except for conditions in which latency is induced, the inabilityof HSV genomes to integrate within host DNA results in susceptibility todegradation of the vector DNA.

In an attempt to circumvent the difficulties inherent in the recombinantHSV vector, defective HSV vectors were employed as gene transfervehicles within the nervous system. The defective HSV vector is aplasmid-based system, whereby a plasmid vector (termed an amplicon) isgenerated which contains the gene of interest and two cis-acting HSVrecognition signals. These are the origin of DNA replication and thecleavage packaging signal. These sequences encode no HSV gene products.In the presence of HSV proteins provided by a helper virus, the ampliconis replicated and packaged into an HSV coat. This vector thereforeexpresses no viral gene products within the recipient cell, andrecombination with or reactivation of latent viruses by the vector islimited due to the minimal amount of HSV DNA sequence present within thedefective HSV vector genome. The major limitation of this system,however, is the inability to eliminate residual helper virus from thedefective vector stock. The helper virus is often a mutant HSV which,like the recombinant vectors, can only replicate under permissiveconditions in tissue culture. The continued presence of mutant helperHSV within the defective vector stock, however, presents problems whichare similar to those enumerated above in regard to the recombinant HSVvector. This would therefore serve to limit the usefulness of thedefective HSV vector for human applications.

For further information on HSV-mediated gene delivery to neurons, seeBreakefield and DeLuca, “Herpes Simplex Virus for Gene Delivery toNeurons,” (1991) New Biologist 3:203-18; Ho and Mocarski (1988)“Beta-Galactosidase as a marker in the herpes simplex virus-infectedmouse,” Virology 167:279-93; Palella, et al (1988) “Herpes SimplexVirus-Mediated human hypoxanthine-guanine phosphoribosyl-transferasegene transfer into neuronal cells,” Molec. & Cell. Biol. 8:457-60;Pallela et al (1988) “Expression of human HPRT mRNA in brains of miceinfected with a recombinant herpes simplex virus-1 vector,” Gene80:137-144; Andersen et al (1992) “Gene transfer into mammalian centralnervous system using the neuron-specific enolase promoter,” Human GeneTherapy 3:487-99; Kaplitt et al (1993) “Molecular alterations in nervecells: Direct manipulation and physiological mediation,” Curr. TopicsNeuroendocrinol. 11:169-191; Spaele and Frenkel (1982) “The HerpesSimplex Virus Amplicon: A New EukaryoticDefective-Virus-Cloning-Amplifying Vector,” Cell 30:295-304 (1982);Kaptitt et al (1991) “Expression of a Functional Foreign Gene in AdultMammalian Brain Following In Vivo Tranfers via a Herpes-Simplex Virus.Type 1 Defective Viral Vector,” Molec. & Cell. Neurosci. 2:320-30;Federoff et al (1992) “Expression of Nerve Growth Factor In Vivo form aDefective Herpes Simplex Virus 1 Vector Prevents Effects of Axotomy onSympathetic Ganglia,” Proc. Nat. Acad. Sci. (USA) 89:1636-40.

While HSV vectors of reduced toxicity and replication ability have beensuggested, they can still mutate to a more dangerous form, or activate alatent virus, and, since the HSV does not integrate, achieving long-termexpression would be difficult.

Adenoviruses

The adenovirus genome consists of about 36 kb of double-stranded DNA.Adenoviruses target airway epithelial cells, but are capable ofinfecting neural cells.

Recombinant adenovirus vectors have been used as gene transfer vehiclesfor non-dividing cells. These vectors are similar to recombinant HSVvectors, since the adenovirus E1a immediate-early gene is removed butmost viral genes are retained. Since the E1a gene is small (roughly 1.5kb) and the adenovirus genome is ⅓ the size of the HSV genome, othernon-essential adenovirus are removed in order to insert a foreign genewithin the adenovirus genome.

In nature, diseases resulting from adenovirus infections are not assevere as those induced by HSV infection, and this is the principaladvantage of recombinant adenovirus vectors compared with HSV vectors.However, retention and expression of many adenovirus genes presentsproblems similar to those described with the HSV vector, particularlythe problem of cytotoxicity to the recipient cell. In addition,recombinant adenovirus vectors often elicit immune responses which mayserve to both limit the effectiveness of vector-mediated gene transferand may provide another means for destruction of transduced cells.Finally, as with the HSV vectors, stability of long-term expression iscurrently unclear since there is no mechanism for specific viralintegration in the genome of non-dividing host cells at high frequency.While theoretically possible, defective adenovirus vectors would bedifficult to make as at least 20% of the Ad genome is required forpackaging (about 27 kb) and vectors this size are difficult to workwith. In contrast, the defective HSV vectors are small plasmids whichreplicate until the correct aggregate size is reached for properpackaging.

For more information on vectors, see Akli et al (1993) “Transfer of aforeign gene into the brain using adenovirus vectors,” Nature Genetics3:224-228; La Salle, et al, “An adenovirus vector for gene transfer intoneurons and glia in the brain,” Science 259:988-90 (1993), Editorial,“Adventures with adenovirus,” 3:1-2 (1993); Neve, “Adenovirus vectorsenter the brain” TIBS 16:251-253 (1993).

Adeno-Associated Virus is a defective parvovirus whose genome isencapsidated as a single-stranded DNA molecule. Strands of plus andminus polarity are both packaged, but in separate virus particles.Although AAV can replicate under special circumstances in the absence ofa helper virus, efficient replication requires coinfection with a helpervirus of the herpesvirus or adenovirus family. In the absence of thehelper virus, AAV establishes a latent infection in which the viralgenome exists as an integrated provirus in the host cell. (No AAV geneexpression is required to establish a latent infection). The integrationof the virus is site-specific (chromosome 19). If a latently infectedcell line is later superinfected with a suitable helper virus, the AAVprovirus is excised and the virus enters the “productive” phase of itslife cycle. However, it has been reported that certain AAV-derivedtransducing vectors are not rescued by adenovirus superinfection.

Although AAV is a human virus, its host range for lytic growth isunusually broad. Cell lines from virtually every mammalian speciestested (including a variety of human, simian, canine, bovine and rodentcell lines) can be productively infected with AAV, provided anappropriate helper virus is used (e.g., canine adenovirus in caninecells). Despite this, no disease has been associated with AAV in eitherhuman or other animal populations, unlike both HSV and adenovirus.

AAV has been isolated as a nonpathogenic coinfecting agent from fecal,ocular and respiratory specimens during acute adenovirus infections, butnot during other illnesses.

Likewise, latent AAV infections have been identified in both human andnonhuman cells. Overall, virus integration appears to have no apparenteffect on cell growth or morphology. See Samulski (1993) Curr. Op. Gen.Devel. 3:74-80.

The genome of AAV-2 is 4,675 bases in length and is flanked by invertedterminal repeat sequences of 145 bases each. These repeats are believedto act as origins for DNA replication.

There are two major open reading frames. The left frame encodes at leastfour non-structural proteins (the Rep group). There are two promoters P5and P19, which control expression of these proteins. As a result ofdifferential splicing, the P5 promoter directs production of proteinsRep 78 and Rep 68, and the P19 promoter, Rep 52 and Rep 40. The Repproteins are believed to be involved in viral DNA replication,trans-activation of transcription from the viral promoters, andrepression of heterologous enhancers and promoters.

The right ORF, controlled by the P40 promoter, encodes the capsidproteins Vp1 (91 kDa), Vp2 (72 kDa) and Vp3 (60 kDa). Vp3 comprises 80%of the virion structure, while Vp1 and Vp2 are minor components. Thereis a polyadenylation site at map unit 95. For the complete sequence ofthe AAV-2 genome, see Vastava et al (1983) J. Virol. 45:555-64.

McLaughlin et al ((1988) J. Virol. 62:1963-73) prepared two AAV vectors:dl 52-91, which retains the AAV rep genes, and dl 3-94, in which all ofthe AAV coding sequences have been deleted. It does, however, retain thetwo 145 base terminal repeats, and an additional 139 bases which containthe AAV polyadenylation signal. Restriction sites were introduced oneither side of the signal.

A foreign gene, encoding neomycin resistance, was inserted into bothvectors. Viral stocks were prepared by complementation with arecombinant AAV genome, which supplied the missing AAV gene products intrans but was itself too large to be packaged.

Unfortunately, the virus stocks were contaminated with wild type AAV(10% in the case of dl 3-94) presumably as a result of homologousrecombination between the defective and the complementing virus.

Samulski et al ((1989) J. Virol. 63:3822-28) developed a method ofproducing recombinant AAV stocks without detectable wild-type helperAAV. Their AAV vector retained only the terminal 191 bases of the AAVchromosome. In the helper AAV, the terminal 191 bases of the AAVchromosome were replaced with adenovirus terminal sequences. Sincesequence homology between the vector and the helper AAV was thusessentially eliminated, no detectable wild-type AAV was generated byhomologous recombination. Moreover, the helper DNA itself was notreplicated and encapsidated because the AAV termini are required forthis process. Thus, in the AAV system, unlike the HSV system, helpervirus could be completely eliminated leaving a helper-free AAV vectorstock.

Muro-Cacho et al ((1992) J. Immunother. 11:231-237) have used AAV-basedvectors for gene transfer into both T- and B-lymphocytes. Walsh et al((1992) Proc. Nat. Acad. Sci. (USA) 89:7257-61) used an AAV vector tointroduce a human gamma globulin gene into human erythroleukemia cells;the gene was expressed. Flothe et al ((1993) J. Biol. Chem. 268:3781-90)delivered the cystic fibrosis transmembrane conductance regulator geneto airway epithelial cells by means of an AAV vector. See also Flotte etal (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-56; Flotte et al (1993)Proc. Nat. Acad. Sci. (USA) 90:10613-17.

SUMMARY OF THE INVENTION

Adeno-associated virus has not been reported to naturally infect anynervous system cells, and AAV-derived vectors have not previously beenused to transfect terminally differentiated, non-dividing cells.Nonetheless, the present invention demonstrates that an adeno-associatedvirus-derived vector may be used to deliver exogenous DNA to cells ofthe postnatal central and/or peripheral nervous system, includingneurons and glia, even though these cells are non-dividing. Specificitymay be achieved by anatomically specific delivery or by tissue specificexpression.

The exogenous DNA preferably comprises a gene which encodes a geneproduct useful in the treatment of a nervous system disorder. This gene,in some embodiments, is operably linked to a promoter specific forparticular cell types or regions within the nervous system. Because theAAV vector is integrated, stable, longterm expression (e.g., for greaterthan seven months) can be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of the AAV vector pAAVlac.

FIG. 2 is a schematic diagram outlining the relationship of the helperplasmid, AAV vector, adenovirus helper, etc.

FIG. 3 shows the effect of intrastriatal AAVth or AAVlac onapomorphine-induced rotational behavior in the rodent model ofParkinson's disease.

FIGS. 4A-F shows the immunohistochemical detection of hTH expressionwithin the caudate nucleus of 6-OHDA lesioned rats following injectionof AAVth. A, Absence of immunostaining in caudate following injection ofAAVlac. No staining was ever observed in AAVlac animals, and stainingwas also always absent from the uninjected caudate from AAVth animals.B,C, TH expression in cells of the caudate nucleus 4 months followinginjection of AAVth. These sections were 30 μm in thickness, whichprevented morphological identification of positive cells. Approximately30 cells are seen at the site of injection (B) and cells are also seen 2mm away from the injection site (C), although fewer cells are present at2 mm. This observation was repeated twice at 4 months followinginjection, while comparable results were obtained from 3 animals at 2months and 2 animals at 1 month following injection. D, TH expression incaudate 1 week following AAVth injection. This section was 7 μm inthickness, revealing the neuronal appearance of the majority of positivecells. 50 positive cells can be seen in this section, which isrepresentative of approximately 50 consecutively positive sectionsobtained from each short-term animal. Fewer cells were observed as faras 280 sections (2 mm) away from the injection site. This result wasrepeated twice at 1 week following injection, and comparable resultswere obtained from 9 animals at 48 hours and 9 animals at 24 hourspost-injection. E,F, Double-label immunocytochemistry demonstratingneuronal TH expression. E, TH expression in a caudate cell (arrow) wasrevealed using a FITC-labelled secondary antibody. F, Neuronalidentification of the TH-expressing cell (arrow) was obtained bysequentially staining the same section with an anti-neurofilamentantibody and visualization with a Texas red-conjugated secondaryantibody. Magnification: A-D, 400× E,F, 630×.

FIG. 5 shows plasmid pAAV-FlagTH-AADC. This bicistronic constructcontains the bicistronic construct with open reading frames fortruncated tyrosine hydroxylase containing the N-terminal Flag epitope(Flag-TH) and aromatic amino acid decarboxylase (AADC). TH convertstyrosine to L-Dopa, and then AADC converts L-Dopa to dopamine. Betweenthe two open reading frames is a sequence allowing ribosome re-entry andinitiation of translation of a second open reading frame downstream froma translational stop codon. This is the internal ribosome entry site(IRES). These are transcribed as a single messenger RNA from the humancytomegalovirus immediate early gene promoter (CMV promoter). At the 3′end of the insert is a signal for polyadenylation of the mRNA derivedfrom the SV40 virus (SV40 polyA). The entire insert is flanked byterminal repeats from the adeno-associated virus (AAV term.), whichpermits replication, excision and packaging of the insert in thepresence of proteins provided by the helper plasmid pAAV/Ad and helperadenovirus. The plasmid also contains standard plasmid sequences whichpermit replication and amplification of the DNA inside a bacterium (ori)and selection of bacterial colonies harboring the plasmid throughresistance to ampicillin (amp). One of the several unique features ofthe AAV vector is that unlike other defective viral vectors, theseplasmid sequences are lost when the DNA between the AAV termini ispackaged.

FIG. 6 shows dopamine release into culture medium following plasmidtransfection in 293T cells. The first group of 4 samples represents 30minutes following addition of tyrosine and tetrahydrobiopterin, whilethe second 4 samples were taken after 60 minutes. Dopamine release wassignificant at 30 minutes, and even higher at 60 minutes, in cellstransfected with pAAV-FlagTH-AADC and given tyrosine andtetrahydrobiopterin (TH/DC+). Cells transfected with this plasmid butnot given the substrate and co-factor (TH/DC−) synthesized negligibleamounts of dopamine at both time points. Controls transfected withpAAVlac, expressing the bacterial lacZ gene, or mock-transfected withphosphate-buffered saline (PBS) produced no detectable amount ofdopamine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The Vector

The vector of the present invention is a derivative of theadeno-associated virus, into which exogenous DNA has been introduced.

While the wild-type adeno-associated virus is already defective, in thatit requires the presence of a helper virus for lytic infection, there isthe possibility that the subject to whom the vector is delivered willharbor a herpesvirus or adenovirus infection which can complement thevector. To guard against this possibility, it is highly preferred thatthe vector be modified to reduce the possibility of rescue. In theory,such modifications can take the form of point mutations to one or moreviral genes, which mutations either prevent expression of the genealtogether, or result in the expression of a modified gene product whichis nonfunctional. However, point mutations are reversible. Consequently,it is preferable that each undesired gene simply be deleted, which hasthe additional advantage of creating more room within the viral packagefor foreign DNA.

It is preferable that all of the viral genes be deleted, or otherwiseinactivated, as in the known AAV vector dl3-94. However, it should beunderstood that a vector retaining one or more AAV genes such as theknown AAV vector dl52-91, may still be useful for gene delivery,although inferior to the preferred vectors.

For propagation of the vector in vitro, susceptible cells areco-transfected with the AAV-derived vector and a suitable AAV-derivedhelper virus or plasmid. Preferably, the vector retains from AAVessentially only the recognition signals for replication and packaging.

It is not necessary that the AAV-derived sequences correspond exactlywith their wild-type prototypes. For example, the AAV vectors of thepresent invention may feature mutated inverted terminal repeats, etc.,provided that the vector can still be replicated and packaged with theassistance of helper virus, and still establish a nonpathogenic latentinfection in target cells.

The vector may further comprise one or more restriction sites into whichforeign DNA may be cloned without interfering with packaging andreplication. Preferably, at least one unique restriction site isprovided. The vector may also comprise one or more marker genes tofacilitate genetic manipulation. Suitable marker genes include, but arenot limited to, the neomycin and hygromycin resistance genes, bacteriallacZ, and the firefly luciferase gene.

The AAV-derived Helper Virus or Plasmid

The AAV-derived helper virus or plasmid may be any virus or plasmidwhich is capable, upon expression of the carried AAV genes, of providingproteins necessary for the replication and packaging of the vector invitro in a suitable host cell, for the purpose of producing vectorstock.

In a preferred embodiment, the helper virus or plasmid is one which hasbeen engineered to reduce the risk of recombination between the helperDNA and the vector DNA. Most desirably, there is essentially no sequencehomology between the AAV sequences of the vector DNA, and the AAVsequences of the helper DNA. For example, the helper DNA may be an AAVin which the AAV inverted terminal repeats are replaced by thecorresponding sequences of another virus, such as adenovirus (e.g.,adenovirus type 5 DNA). See Samulski et al J. Virol. 63:3822-28.

Alternatively, in another preferred embodiment, helper adenovirus may beremoved by heat inactivation at 56° C. for 30 minutes, or separated frompackaged AAV vectors by centrifugation in a cesium chloride gradient.

Exogenous DNA

Basic procedures for constructing recombinant DNA and RNA molecules inaccordance with the present invention are disclosed by Sambrook, J. etal, In: Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989), which reference isherein incorporated by reference.

The “exogenous DNA” of the present invention should be exogenous to bothAAV and to the target cell. The DNA may be synthetic DNA, complementaryDNA, genomic DNA, or a combination thereof. The DNA may be of anysequence or length, provided that it may be incorporated into the vectorand delivered to target cells. Typically, because of the packaginglimitations of AAV, the exogenous DNA will have a length of about10-5,000 bases. Preferably, the DNA is 100 to 4,000 bases.

The present invention may be used for gene therapy of anygenetically-based or—acquired nervous system disorder. An individual maybe in need of gene therapy because, as a result of one or more mutationsin the regulatory region and/or the coding sequence of one or moregenes, a particular gene product is inappropriately expressed, e.g., hasan incorrect amino acid sequence, or is expressed in the wrong tissuesor at the wrong times, is underexpressed or overexpressed. Therefore,DNA delivered to that individual may be considered exogenous even thoughit is identical to a gene native to that individual's species, providedit differs in the regulatory or coding region from the cognate gene ofthe individual to whom it is delivered, and therefore encodes adifferent gene product or is expressed to a different degree and/or indifferent cells, under at least some conditions.

Parkinson's Disease

Current approaches to Parkinson's Disease (PD) are based on facilitatingdopaminergic neurotransmission in the caudate-putamen (CP). The mainstayof treatment is oral L-Dopa (and a peripheral decarboxylase inhibitor),which is converted to dopamine by endogenous AADC in the denervated CP.Alternative pharmacological approaches include direct dopamine agonistsincluding bromocriptine and apomorphine, as well as dopaminemetabolizing enzyme (e.g. monoamine oxidase) inhibitors (MAOI), e.g.deprenyl. Although these treatments have made a significant improvementin the short term quality of life of PD patients, the disease progresseswith all patients ultimately becoming refractory to oral treatment over5 to 10 years.

Alternative investigative strategies have included fetal and adrenaltransplantation, which although showing promise in animal models of PD,have had marginal efficacy in the human. Moreover, these transplantationapproaches disturb the neuronal environment, abnormal sprouting andsynapse formation occurs and the transplanted cells may induce an immuneresponse as well as being targeted for the same underlyingneurodegeneration responsible for the PD. A further implantationstrategy includes a polymer or encapsulation device in which cellseither dopamine-producing (e.g. PC 12) or genetically engineered cells(fibroblasts or neuronal cell lines which have been transduced,typically by retroviruses, to express tyrosine hydroxylase enzyme[THE]). These implants also disturb the neuronal circuitry, createsignificant injury in view of the size of the implant and moreovergenerate high local concentrations of dopamine to potentially toxicconcentrations.

A more elegant approach is to use a viral vector to transduce neurons invivo. We have previously established that an HSV-1 vector expressing THEcan be stereotactically implanted into the denervated striatum of a ratmodel of PD and obtain biochemical and behavioral recovery extending atleast one year (During et al, submitted).

The current invention has the significant advantages over the HSV-1defective vector approach. Specifically, the reversion frequency of thedefective HSV-1 virus is approximately 10⁻⁵, and with the amounts ofvirus needed for in vivo studies sufficient wild type herpes infectionoccurs to result in toxicity and the death of experimental animals.Furthermore, although expression in the first two weeks is high, thelevel of vector gene expression beyond 2 weeks is reduced, perhaps to5-20% of the initial expression.

The current invention also provides a major advantage over approacheswhich limit expression to THE. In PD (and the denervated striatum inanimal models of PD) not only is the enzyme THE decreased by 80-100%,but the second enzyme in the dopamine biosynthetic pathway, AADC, isalso decreased by approximately 85%. As L-Dopa per se does not restoredopaminergic activity and behavioral recovery in PD, production ofdopamine in the denervated striatum where THE is restored may becomelimited by the activity of AADC. The ability for cells to acquire thefull dopaminergic phenotype (by expressing both THE and AADC) is likelyto be more effective. In support of this claim, one group has shown thatwhen comparing animals transplanted with genetically engineered cellsexpressing only THE to animals implanted with both THE and AADCexpressing transplants, the latter group had better recovery.

Therefore, in one embodiment, the vectors of the present invention areused to deliver the gene for tyrosine hydroxylase (Genbank HUMTHX,Accession No. M17589) to brain cells. Preferably, the gene for aromaticamino-acid decarboxylase (Genbank HUMDDC, Accession No. M76180) issimilarly delivered, by the same or a separate vector.

The above description of transducing striatal cells in vivo to adopaminergic phenotype is the first step in a gene therapy approach toPD. However, PD becomes symptomatic when 80% of the dopamine neuronshave been lost. Degeneration is progressive and with further denervationpatients become increasingly refractive to all current therapies andexhibit “On-Off” phenomenon with increasing freezing and completeimmobility. Transducing striatal cells with a viral vector to expressdopamine synthesizing enzymes is purely a palliative approach and theunderlying disease process will continue unabated. To this end vectorshave been constructed expressing “neuroprotective or neurotrophic”factors to prevent further degeneration of dopaminergic neurons andpromote regeneration. This approach includes the most specificneurotrophic factor for mesencephalic dopaminergic neurons identified todate, glial-derived neurotrophic factor (GDNF). Other neurotrophicfactors of the NGF family have previously been expressed from HSV-1vectors and shown to have neuroprotective effects (Federoff et al. Theseneurotrophic factors appear to act through tyrosine kinase receptors toprevent apoptosis or programmed cell death (PCD). As the proto-oncogenebcl-2 can prevent neuronal PCD in vitro, an AAV vector has beenconstructed expressing bcl-2 to prevent PCD in vivo. This vector mighttherefore be considered for any neuronal degeneration in the brainincluding ischemia, epilepsy or brain trauma where secondary neuronalinjury occurs via PCD.

Therefore, gene therapy for PD could involve delivery, by AAV vectors,of the gene for GDNF (Genbank HUMGDNF02; Accession No. L19063),brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)(EMBL HSNGF2; Accession No. X53655, and/or other members of theneurotrophin factor family including neurotrophin (NT)-3 (GenbankHUMBDNF; Accession No. M37762) and NT-4 (Genbank HUMPPNT4P; AccessionNo. M86528).

Recent evidence strongly implicates oxidative stress in the substantianigra as a primary determinant of the progressive neuronal loss.Specifically, iron appears to be concentrated in the nigra of PDpatients and studies have shown iron binding to neuromelanin of thenigra cells to generate free radicals. An antioxidant strategy to PD hastherefore been proposed. The key enzymes which reduce free radicalgeneration and/or scavenge free radicals are superoxide dismutase (SOD),catalase and glutathione peroxidase (GPO). Although mutations oralterations in expression of these enzymes has not yet been determinedin PD, the increased expression of these enzymes in the nigrostriataldopaminergic neurons will increase their ability to withstand oxidativestress. Therefore, an AAV vector has been made expressing SOD. This SODexpressing vector is also of interest in the treatment of amyotrophiclateral sclerosis (ALS) in which in the familial form is associated withmutations in the SOD-1 gene.

Therefore, it may be desirable to use AAV vectors to deliver the genesfor superoxide dismutase (SOD1 or SOD2) (GenBank HUMCUZNDI; AccessionNo. M12367; for SOD-1, EMBL HSSOD2G, Accession No. X65965 for SOD-2),catalase (EMBL HSCATR, Accession No. X04076), and/or glutathioneperoxidase (MBL HSGSHPX, Accession No. Y00433).

Epilepsy

Complex partial seizures and specifically temporal lobe epilepsy (TLE)is one of the most refractory forms of epilepsy. Although antiepilepticdrugs (AED) will control seizures in some patients, 40% will remainuncontrolled despite polyAED therapy. The current approach for thesepatients is to undergo a phased evaluation for consideration ofresective surgery. Typically, one temporal lobe is defined as the siteof seizure origin (the epileptogenic region) and the medial temporallobe including the anterior hippocampus is resected. TLE is not agenetic disease and there is no established aetiology, however thedisease results from an imbalance of excitation to inhibition withinterventions enhancing excitation or blocking inhibition producingseizures and conversely the antagonism of excitation and enhancing ofinhibition has an antiepileptic effect. One gene therapy approach to TLEis to improve inhibition. To this end, the adenosine A-1 receptor(GenBank S56143; Accession S56143) cDNA has been inserted into the AAVvector. As adenosine has been shown to be the brain's naturalanticonvulsant (acting through A-1 receptors) and levels of the receptorare decreased in the epileptogenic region, this strategy is likely toenhance inhibition and prevent seizures.

Alternative strategies to increase inhibition include the insertion ofgenes whose expression would enhance GABAergic activity. These geneswould include GABA_(A) receptor isoforms and the GABA synthesizingenzyme, GAD.

One related approach to increase inhibition is to increase expression ofion channels which alter neuronal excitability, specifically activitydependent channels including calcium-activated potassium channels andATP-sensitive potassium channels.

A complementary approach is to express an antisense to excitatoryreceptors specifically glutamate receptors including NMDAR's, mGluR'sand ionotropic glutamate receptors including both AMPA and kainate. Aswe have shown that expression of GluR6 using a HSV-1 vector inducesepilepsy, it is reasonable to predict that a vector expressing antisenseto these receptor types may inhibit seizures.

Therefore, for the treatment of epilepsy, genes encoding adenosine A-1receptor (GenBank S56143; Accession S56143, glutamate decarboxylase(GenBank S61898; Accession S61898), GABA-A receptor isoforms (EMBLHSGABAAA1; Accession X14766), calcium-dependent potassium channels(GenBank DROKCHAN, Accession M96840) and/or ATP-sensitive potassiumchannels (Ho, et al 1993 Nature 362:31-8) may be delivered by AAVvectors.

Brain Tumors

Brain tumors are intractable and usually lethal diseases which largelyaffect children and adults in middle age. Currently, there are no knowncauses of most brain tumors and treatment modalities have been largelyineffectual. There have been two recent strategies using gene therapy aspossible approaches for brain tumor therapy. In the first approach, amutant HSV has been used which can replicate only in dividing cells.This should result in destruction of dividing tumor cells while sparingnon-dividing cells in the healthy brain. Although there has been someexperimental evidence supporting the fact that these viruses do reducethe rate of brain tumor growth in experimental animals, results havebeen variable and not as impressive as would be desired. There have beenno human trials of this method.

The second approach involves the insertion of the thymidine kinase (TK)gene from herpes simplex virus type 1 into a replication-deficientretroviral vector. The retroviral vector only transferred the TK geneinto dividing tumor cells, but could not transfer genes either intonon-dividing tumor cells or healthy brain tissue.

It was found in tissue culture and in animal tumors that cellstransduced with TK via the retroviral vector would become susceptible tocytotoxicity by the drug ganciclovir. TK phosphorylates ganciclovir, andthe phosphorylated form disrupts DNA replication and thereby killsdividing cells. It was also found that nearby dividing cells which werenot transduced with TK could also be killed. This was called thebystander effect, as it is believed that some phosphorylated drug canexit the transduced cell and enter nearby, non-transduced cells via gapjunctions. Non-dividing cells are unaffected even by activated drug.This approach to tumor therapy is currently in clinical trials at theNIH.

Since results with this therapy have also been variable in animalstudies, more recent experiments have shown that use of areplication-competent retroviral vector can improve the response inanimals. This approach would not be applicable to human disease,however, due to the extreme danger of placing large amounts of wild-typeretroviruses directly into human patients. This has never been permittedin human patients previously due to the potential for cytotoxicity andinduction of de novo tumors following random integration of wild-typeretroviruses into the genome of the recipient cell.

This embodiment of the current invention envisions a significantimprovement over these previous studies. Insertion of the TK gene (EMBLHEHSV1TK, Accession X03764; EMBL HEHS07, Accession V00466), into the AAVvector should permit transduction of genes into dividing tumor cellswith efficiencies that are at least equal to retroviral vectors, andpossibly with greater efficiency (which has been observed in comparisonsof AAV and defective HSV vectors in rat brain). Unlike retroviruses,however, AAV vectors will also transfer the TK gene into slowly dividingor non-dividing cells within tumors as well as non-dividing normalcells. This could have two significant advantages compared withretroviral vectors.

First, the ability to phosphorylate drugs within non-dividing tumorcells and normal cells should create a greater pool of activated drugwithin the tumor. Given the observation of the bystander effect,non-dividing tumor cells containing the HSV TK gene should phosphorylatethe drug and this could then enter a nearby dividing cell which may nothave been transduced with the viral gene. Thus, a non-dividing cellcould permit destruction of a nearby, non-transduced cell, even thoughthe transduced, non-dividing cell would not be adversely effected. Inthis manner, a greater population of dividing cells would be destroyed.

The second advantage is the ability of AAV vectors to integrate innon-dividing cells. If a retrovirus enters a non-dividing cell, reversetranscription does not occur and the vector is lost. When the AAV vectorenters a non-dividing tumor cell, however, the vector should integrateinto the host genome. Thus, if that tumor cell then re-enters celldivision, the TK gene should be retained in that cell and all progeny.This should then render such previously quiescent tumor cellssusceptible to destruction by ganciclovir or an analog. Since retroviralvectors are lost in non-dividing cells, and other DNA viral vectors donot reliably integrate within the host genome, the ability to retain theTK gene if a quiescent cell begins division is a property unique to theAAV vector. Finally, it should be reiterated that integration of the AAVvector should not result in disruption or abnormal regulation of hostgenes, and that transduction of normal non-dividing cells with TK shouldnot have any adverse effects, since it is the subsequent activation ofthe drug by TK which blocks DNA replication and this only results indestruction of dividing cells. Thus, this embodiment of the inventionprovides substantial improvements over previous drug-susceptibilitytumor treatment strategies.

Target Cells

The target cells of the vectors of the present invention are cells ofthe central or peripheral nervous systems of a mammal. In oneembodiment, the cells are cells cultured in vitro.

In another embodiment, the cells are part of a living mammal at the timethe vector is delivered of the cell. The mammal may be at any stage ofdevelopment at the time of delivery, e.g., embryonic, fetal, infantile,juvenile or adult.

The vector may be delivered to cells of the central nervous system,cells of the peripheral nervous system, or both. When the vector isdelivered to the cells of the central nervous system, it may bedelivered to cells of the spinal cord, brainstem (medulla, pons, andmidbrain), cerebellum, diencephalon (thalamus, hypothalamus),telencephalon (corpus striatum, cerebral cortex, or, within the cortex,the occipital, temporal, parietal or frontal lobes), or combinationsthereof. Similarly, within the peripheral nervous system, it may bedelivered to cells of the sensory and/or effector pathways.

To deliver the vector specifically to a particular region of the centralnervous system, it may be administered by stereotaxic microinjection, asexemplified in Example 2. For example, on the day of surgery, patientswill have the stereotactic frame base fixed in place (screwed into theskull). The brain with stereotactic frame base (MRI-compatible withfiducial markings) will be imaged using high resolution MRI. The MRIimages will then be transferred to a computer which runs stereotacticsoftware. A series of coronal, sagittal and axial images will be used todetermine the target (site of AAV vector injection) and trajectory. Thesoftware directly translates the trajectory into 3 dimensionalcoordinates appropriate for the stereotactic frame. Burr holes aredrilled above the entry site and the stereotactic apparatus positionedwith the needle implanted at the given depth. The AAV vector will thenbe injected at the target sites. Since the AAV vector will integrateinto the target cells, rather than producing viral particles, thesubsequent spread of the vector will be minor, and mainly a function ofpassive diffusion from the site of injection, prior to integration. Thedegree of diffusion may be controlled by adjusting the ratio of vectorto fluid carrier.

If a more widespread distribution of the vector across the CNS isdesirable, it may be injected into the cerebrospinal fluid, e.g., bylumbar puncture.

To direct the vector to the peripheral nervous system, it may beinjected into the spinal cord, or if more limited PNS distribution issought, into the peripheral ganglia, or the flesh (subcutaneously orintramuscularly) of the body part of interest.

In certain situations the vector will be administered via anintravascular approach. For example, the vector will be administeredintra-arterially (carotid) in situations where the blood-brain barrieris disturbed. Such conditions include cerebral infarcts (strokes) aswell as some brain tumors. Moreover, for more global delivery, thevector will be administered during the “opening” of the blood-brainbarrier achieved by infusion of hypertonic solutions including mannitol.Of course, with intravenous delivery, the user must be able to toleratethe delivery of the vector to cells other than those of the nervoussystem.

The vector may also be delivered intracerebroventricularly and/orintrathecally, for specific applications, including vectors expressingsuperoxide dismutase and neurotrophic factors for amyotrophic lateralsclerosis and Alzheimer's Disease and genes encoding enzymes ofneurogenetic diseases e.g., Tay Sachs and Lesch-Nyan disease.

Additional routes of administration will be local application of thevector under direct visualization, e.g., superficial corticalapplication, or other non-stereotactic application.

For targeting the vector to a particular type of cell, e.g., a neuron,it is necessary to associate the vector with a homing agent that bindsspecifically to a surface receptor of the cell. Thus, the vector may beconjugated to a ligand (e.g., enkephalin) for which certain nervoussystem cells have receptors. The conjugation may be covalent, e.g., acrosslinking agent such as glutaraldehyde, or noncovalent, e.g., thebinding of an avidinated ligand to a biotinylated vector. Another formof covalent conjugation is provided by engineering the helper virus usedto prepare the vector stock so that one of the encoded coat proteins isa chimera of a native AAV coat protein and a peptide or protein ligand,such that the ligand is exposed on the surface.

Whatever the form of conjugation, it is necessary that it notsubstantially interfere either with the integration of the AAV vector,or with the binding of the ligand to the cellular receptor.

The target cells may be human cells, or cells of other mammals,especially nonhuman primates and mammals of the orders Rodenta (mice,rats, rabbit, hamsters), Carnivora (cats, dogs), and Arteriodactyla(cows, pigs, sheep, goats, horses).

Gene Expression

When the exogenous DNA comprises an expressible gene the gene may be onewhich occurs in nature, a non-naturally occurring gene which nonethelessencodes a naturally occurring polypeptide, or a gene which encodes arecognizable mutant of such a polypeptide. It may also encode an mRNAwhich will be “antisense” to a DNA found or an mRNA normally transcribedin the host cell, but which antisense RNA is not itself translatableinto a functional protein.

The precise nature of regulatory regions needed for gene expression mayvary from organism to organism, but in general include a promoter whichdirects the initiation of RNA transcription. Such regions may includethose 5′-non-coding sequences involved with initiation of transcriptionsuch as the TATA box. The promoter may be constitutive or regulatable.Constitutive promoters are those which cause an operably linked gene tobe expressed essentially at all times. Regulatable promoters are thosewhich can be activated or deactivated. Regulatable promoters includeinducible promoters, which are usually “off” but which may be induced toturn “on”, and “repressible” promoters, which are usually “on” but maybe turned off. Many different regulators are known, includingtemperature, hormones, heavy metals, the product of the natively linedgene, and regulatory proteins. These distinctions are not absolute; aconstitutive promoter may be regulatable to some degree.

The regulatability of a promoter may be associated with a particulargenetic element, often called an “operator”, to which an inducer orrepressor binds. The operator may be modified to alter its regulation.Hybrid promoters may be constructed in which the operator of onepromoter is transferred into another.

The promoter may be an “ubiquitous” promoter active in essentially allcells of the host organism, e.g., the beta-actin or optomegaloviruspromoters, or it may be a promoter whose expression is more or lessspecific to the target cells. Preferably, the tissue-specific promotersare essentially not active outside the nervous system, and the activityof the promoter optionally may be higher in some components of thenervous system than in others.

Thus, the promoter may be one which is active primarily in the centralnervous system, or primarily in the peripheral nervous system, or it maybe significantly active in both. If it is active in the CNS, it may bespecific for the spinal cord, the brainstem (medulla, pons, midbrain, orcombinations thereof), the cerebellum, the diencephalon (thalamus and/orhypothalamus), the telencephalon (the corpus striatum and/or thecerebral cortex, and, if the latter, the occipital, temporal, parietaland/or frontal lobes), or combinations thereof. The specificity may beabsolute or relative.

Similarly, the promoter may be specific for particular cell types, suchas neurons or glial cells in the case of the CNS, or particularreceptors or effectors in the case of the PNS. If it is active in glialcells, it may be specific for astrocytes, oligodendrocytes, ependymalcells, Schwann cells, or microglia. If it is active in neurons, it maybe specific for particular types of neurons, e.g., motor neurons,sensory neurons, or interneurons.

In general, to find a tissue-specific promoter, one identifies a proteinwhich is expressed only (or primarily) in that tissue, and then isolatesthe gene encoding that protein. (The gene may be a normal cellular gene,or a viral gene of a virus which infects that cell). The promoter ofthat gene is likely to retain the desired tissue-specific activity whenlinked to another gene.

The tissue specificity of a promoter may be associated with a particulargenetic element, which may be modified, or transferred into a secondpromoter.

Control of expression to specific cell types will be obtained using geneexpression control elements. Specifically, one may use these approaches:

(1) Expression in all cell types:

Both strong viral (e.g.immediate early CMV, available on plasmid pCDNA1from Invitrogen, Inc., San Diego, Calif.) and relatively non-specificcellular promoters (e.g., β-actin, Genbank HUMACTBET, K00790) may beused to direct expression in all cell types.

(2) Neuronal specific expression:

Approaches will include the use of neuron specific promoters e.g.,neuron specific enolase (EMBL HSENO2, X51956), AADC, neurofilament(Genbank HUMNFL, L04147), synapsin (Genbank HUMSYNIB, M55301), andserotonin receptor (Genbank S62283), promoters, as well as thecombination of more broadly active promoters together with silencerelements which restrict expression to neurons.

(3) Glial specific expression:

Approaches will include use of the glial fibrillary acidic protein(GFAP) promoter (Genbank HUMGFAP, J04569), S100 promoter (GenbankHUMS100AS, M65210), and glutamine synthase (EMBL HSGLUS, X59834)promoter.

(4) Expression may be restricted to specific neuronal subpopulationsusing the following genetic elements:

Peptidergic promoters: e.g., enkephalin (Genbank HUMENKPH1, K00488),prodynorphin, somatostatin (Genbank RATSOMG, J00787; Genbank HUMSOMI,J00306); monoaminergic promoters: tyrosine hydroxylase (Genbank M23597),dopamine β-hydroxylase (Genbank RATDBHDR, M96011), PNMT (EMBL HSPNMTB,X52730); for cholinergic neurons: choline acetyltransferase promoter(Genbank HUMCHAT1, M89915; EMBL HSCHAT, X56585).

For the gene to be expressible, the coding sequence must be operablylinked to a promoter sequence functional in the target cell. A promoterregion would be operably linked to a coding sequence if the promoterwere positioned so that, when the promoter was activated, the codingsequence was transcribed. The coding sequences are operably linked ifthe linkage does not cause an error in the reading of the downstreamsequence. In order to be “operably linked” it is not necessary that twosequences be immediately adjacent to one another.

If desired, the non-coding region 3′ to the gene sequence coding for thedesired RNA product may be obtained. This region may be retained for itstranscriptional termination regulatory sequences, such as those whichprovide for termination and polyadenlylation. Thus, by retaining the3′-region naturally contiguous to the coding sequence, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals natively associated with the codingsequence are not satisfactorily functional in the expression host cell,then a different 3′ region, functional in the host cell, may besubstituted.

An “expression vector” is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA molecule which has been cloned into thevector and of thereby producing an RNA or protein product encoded by anexpressible gene provided by said DNA. Expression of the clonedsequences occurs when the expression vector is introduced into anappropriate host cell. If a prokaryotic expression vector is employed,then the appropriate host cell would be any prokaryotic cell capable ofexpressing the cloned sequences. Similarly, when a eukaryotic expressionvector is employed, e.g., for genetic manipulation prior to genedelivery, then the appropriate host cell would be any eukaryotic cellcapable of expressing the cloned sequences.

In addition to or instead of an expressible gene, the nucleic acid maycomprise sequences homologous to genetic material of the target cell,whereby it may insert itself into the genome by homologousrecombination, thereby displacing a coding or control sequence of a geneor deleting a gene altogether, provided that these sequences do notsubstantially interfere with integration of AAV.

In another embodiment, the nucleic acid molecule is “antisense” to agenomic or other DNA sequence of the target organism (including virusesand other pathogens) or to a messenger RNA transcribed in cells of theorganisms, which hybridizes sufficiently thereto to inhibit thetranscription of the target genomic DNA or the translation of the targetmessenger RNA. The efficiency of such hybridization is a function of thelength and structure of the hybridizing sequences. The longer thesequence and the closer the complementarily to perfection, the strongerthe interaction. As the number of base pair mismatches increases, thehybridization efficiency will fall off. Furthermore, the GC content ofthe packaging sequence DNA or the antisense RNA will also affect thehybridization efficiency due to the additional hydrogen bond present ina GC base pair compared to an AT (or AU) base pair. Thus, a targetsequence richer in GC content is preferable as a target.

It is desirable to avoid antisense sequences which would form secondarystructure due to intramolecular hybridization, since this would renderthe antisense nucleic acid less active or inactive for its intendedpurpose. One of ordinary skill in the art will readily appreciatewhether a sequence has a tendency to form a secondary structure.Secondary structures may be avoided by selecting a different targetsequence.

In still another embodiment, the gene encodes a ribozyme, i.e., an RNAwith a desirable enzymatic activity.

SUMMARY OF EXAMPLES

Current approaches to transfer genes into the nervous system employeither recombinant viral vectors which retain viral genes or defectivevectors containing residual and potentially dangerous helper viruses.Adeno-associated viral (AAV) vectors are non-pathogenic integrating DNAvectors in which all viral genes are removed (96% of the viral genome)and helper virus is completely eliminated. An AAV vector expressingβ-galactosidase was stereotactically injected into rat brain regionsincluding striatum, hippocampus and substantia nigra. Vector DNA andtransduced gene expression was detected from 1 day to 3 monthspost-injection. A second vector expressing human tyrosine hydroxylase(TH) was generated. This vector (AAVth) was injected into the denervatedstriatum of unilateral 6-hydroxydopamine-lesioned rats and THimmunoreactivity was obtained in striatal cells, including both glia andneurons, to 4 months. There was no evidence of pathology or toxicity inany animal treated with AAV vectors. Initial data indicates that THtransduction in the striatum via an AAV vector yields significantbehavioral recovery in lesioned rats compared with AAVlac controls.

MATERIALS AND METHODS

Plasmids: Plasmid pSub201 (Samulski et al (1989) J. Virol. 63:3822-28)was digested with XbaI to remove nearly the entire AAV genome, leavingonly the terminal repeats. A CMV promoter-lacZ gene-SV40 polyA signalcassette was isolated from plasmid pHCL (Kaplitt et al (1991) Mol. Cell.Neurosci. 2:320-30) by digestion with SpeI and XbaI, and this wasinserted into XbaI-digested pSub201 to create pAAVlac. A second plasmidwas created (pAAV-CMV-polyA) by digestion of pAAVlac with HindIII andXbaI to remove the lacZ gene and polyA signal, followed by insertion ofa HindIII-XbaI fragment from pREP4 (Invitrogen), containing a polylinkerand SV40 polyA signal. This plasmid was then digested with HindIII andBamHI, followed by insertion of a human tyrosine hydroxylase (hTH) cDNA(O'Malley et al (1987) Biochemistry 26:6910-14) in order to createpAAVth.

Creation of Defective Viral Vectors: In order to create AAV vectors,plasmids (pAAVlac or pAAVth) were transfected via the calcium phosphatemethod (Graham et al (1973) Virology 52:496-67) into 293T cells, avariant of 293 cells (Graham et al (1977) J. Gen. Virol. 36:59-74),(obtained from D. Baltimore) which constitutively express both theadenovirus E1a protein and the SV40 T antigen. The vector plasmids wereco-transfected along with the helper plasmid pAd8, which providesnecessary replication and structural proteins. The next day, cells wereinfected with adenovirus strain dl309 (Jones and Shenk (1978) Cell13:181-88) (obtained from Thomas Shenk, Princeton University). Followingfull cytopathic effect, virus was harvested by multiple freeze/thawcycles. Viral stocks were then heated to 56° C. for 30 minutes in orderto inactivate residual adenovirus (Samulski et al 1989). Vector titerswere obtained by histochemical assay for X-gal (Kaplitt et al (1991)Mol. Cell. Neurosci. 2:320-30) or immunocytochemical identification ofhTH expression in 293T cells infected with serial dilutions of thevector stock, using a monoclonal anti-hTH antibody (Boehringer Mannheim)and the ABC elite detection system (Vector Labs).

Immunocytochemistry and X-Gal Histochemistry: For analysis of brainsections from animals injected with AAVlac, tissues were fixed byintracardiac perfusion with 2% paraformaldehyde/5 mM EGTA/2 mM MgCl₂ in0.1 M HEPES (pH 7.3). The addition of EGTA eliminates any backgroundstaining due to endogenous cellular enzymes. Tissue culture cells werefixed with 2% formaldehyde/0.2% glutaraldehyde in PBS (pH 7.2). X-galhistochemistry for detection of β-galactosidase expression was performedas described previously.

In Situ PCR: Brain sections were placed in detergent buffer (0.01%sodium deoxycholate/0.02% NP-40 in PBS) for 1 hour. Following PBS wash,sections were dehydrated in alcohol and 200 μl of PCR reaction bufferwas added to each slide (PCR reaction buffer: 1×PCR buffer/1 μM eachprimer/1M MgCl₂/10 μl digoxigenin-dUTP (Boehringer)). Primers specificfor the lacZ gene are as follows:

N-terminal-5′ to 3′ CCGACTGATGCCTTCTGAACAA (SEQ ID NO: 1) (referred toas lacZ 182) The downstream primer again 5′ to 3′ GACGACAGTATCGGCCTCAGGA(SEQ ID NO:2) (lacZ 560).

Slides were coverslipped and coverslips were anchored on one side withnail polish. Slides were placed on aluminum foil on the block of athermal cycler, and the temperature was raised to 82° C. Coverslips wereraised, 2 μl of enzyme mix (1×PCR buffer/2U/ml Taq) was added to eachslide and coverslips were dropped. Slides were covered in mineral oil,and the following profile was run: 35 cycles of 2 minutes, 55° C.; 2minutes, 72° C.; 2 minutes, 94° C. Slides were placed in xylene toremove the mineral oil, and sections were re-hydrated. PCR product wasdetected in situ with an alkaline phosphatase-labelled anti-digoxigeninantibody, according to the manufacturer's instructions.

Animals and Tissue Preparation: Male Sprague-Dawley rats were used inall studies. Animals were treated according to the NIH Guidelines forAnimal Care and Use. For surgical procedures, animals were anesthetizedwith a mixture of enflurane and NO₂. Stereotaxic microinjection was usedfor all brain region injections, and coordinates were determinedaccording to the atlas of Paxinos and Watson, The Rat Brain inStereotaxic Coordinates, (Academic Press, Sydney, Australia: 1982).Tissue for immunocytochemistry was removed and quickly frozen mountingmedium. 5 μm sections were taken with a cryostat, and sections werefixed in buffered formalin. Tissue for X-gal histochemistry was preparedas described above.

Unilateral Substantia Nigra Lesioning: Unilateral nigral lesions weregenerated using the method of Perese et al (1989) Brain Res. 494:285-93,as previously described, During et al (1992) Exp. Neurol. 115:193-99. Inbrief, male Sprague Dawley rats 290-310 grams were anesthetized withxylazine/ketamine and placed in a Kopf stereotactic frame. The skull wasexposed and burr holes drilled above the left substantia nigra, Lambda+3.5, L 2.15. Freshly made 6-hydroxydopamine (4 μg in 2 μl of 0.1%ascorbic acid in PBS) was loaded into a Hamilton syringe which waslowered into two sites over 2 minutes. The coordinates of the medialsite was lateral 1.9 and ventral 7.1 mm with the needle bevel facingrostrally, whereas the lateral site is 2.3 mm lateral and 6.8 mm in thedorsal ventral plane with the needle bevel oriented laterally (Graham etal. (1977) J. Gen. Virol. 36:59-74). At each site 2 μl was injected over5 minutes and the needle left in place for a further 5 minutes beforebeing withdrawn over an additional 5 minutes.

Behavioral Testing: Rats were tested 10-16 days following the 6-OHDAinjections. They were placed in a hemispherical rotameter and the totalnumber of complete body turns was recorded from 15-20 minutes followingthe administration of apomorphine (1 mg/kg) as described by Hefti et al.(1980), Brain Res., 195:123-27. A minimum of three tests separated by atleast 2 weeks was used to generate a basal rotation rate. Animals whichconsistently exhibited stable (less than 25% variation) asymmetricalrotational behavior of greater than 10 turns per minute were randomlyselected for either AAVlac or AAVth injection.

Stereotactic injection of AAVlac or AAVth in 6-OHDA lesioned rats: Ratsmeeting the above behavioral criteria of a near complete lesion wereanesthetized with ketamine/xylazine (70 mg/7 mg per kg) and placed in akopf stereotactic frame. The skull was exposed and holes drilled abovethe denervated striatum (left) at Paxinos & Watson coordinates of AP0.2, L 2.6 and AP 1.5, L 2.0 and L 3.0. Either AAVlac of AAVth wasinjected slowly using a Hamilton syringe into each of three sites at aDV depth of 5 mm. Each injection volume was 2 μl. Rats were tested forapomorphine-induced rotational behavior at one and two months followingsurgery.

TH, NF and GFAP Immunocytochemistry: For immunohistochemical (IHC)analysis of brain sections, rats were deeply anesthetized with chloralhydrate and followed by intracardiac perfusion with IM PBS (pH=7.3)followed by 4% paraformaldehyde (PF). Brains were removed and post-fixed(3-4 hours) in 4% PF followed by ascending sucrose solutions (10/15/30%in PBS) as cryoprotectant. Sections (7-30 mm) were cut in a cryostat(Reichert-Jung) and mounted on polylysine-coated slides. We used adouble labelling protocol for the co-detection of TH positive cells inspecific neuronal or glial subpopulations. Sections were initiallyincubated in blocking buffer (5% Goat Serum (GS)/5% Normal Horse Serum(NHS) in 1 M Phosphate Buffer Saline (PBS). Sections were than incubatedin primary antibodies diluted in blocking buffer (mouse anti-TH[Boehringer Mannheim, 1:200], mouse anti-NF [Sigma, 1:400], rabbitanti-TH [Chemicon International, 1:3000]rabbit anti-GFAP [gift fromDept. of Pathology, Memorial Hospital, 1:800]), at room temperature (2-4hours), rinsed in blocking buffer and incubated in secondary antibodies(mouse anti-texas red [vector, 1:75 or biotinylated rabbit anti-IgG[vector, 1:400]) at room temperature (1 hour). Sections were washed inPBS and incubated at room temperature (1 hour) in avidin-neutralite FITC[Molecular Probes Inc, 1:400]. Slides were coverslipped withPBS/Glycerol (0.05:1) and kept at −20° C.

EXAMPLE 1 Creation of an Adeno-associated Virus (AAV) Vector for GeneTransfer Into Brain

The bacterial lacZ gene was inserted into plasmid psub20l (Samulski etal (1989) J. Virol. 63:3822-28) between the termini of the AAV genome.These termini contain the recognition signals for cleavage and packaginginto an AAV vector. The lacZ gene encodes the bacterial enzymeβ-galactosidase; which produces an insoluble blue precipitate uponreaction with the appropriate substrate. The human cytomegalovirus (CMV)immediate-early promoter was used to direct gene expression, and an SV40polyadenylation signal was placed downstream of the lacZ gene (FIG. 1).Cells were transfected with pAAVlac and a second plasmid, pAd8 (Samulskiet al. 1989), which provides AAV structural proteins but lacks AAVtermini and thus cannot package into virus. Co-transfected cells werethen infected with adenovirus type 5 (Jones and Shenk (1978) Cell13:181-188) to provide remaining replication and packaging machinery(FIG. 2). The resulting stock consisted of packaged AAV-lac vectors(AAVlac) and progeny helper adenovirus; helper virus was then eliminatedby heating at 56° C. for 30 minutes. The complete elimination ofadenovirus was confirmed by the inability to detect any viral plaques incultured cells 1 week following infection with this viral stock. AAVvectors were then titered by infection of cultured 293 cells,histochemical staining for β-galactosidase expression and counting ofblue cells. There was no difference in the number of cells observed at 1and 5 days following infection, demonstrating an absence of vectorreplication and spread. When the process was repeated using a lacZplasmid without the AAV recognition signals, nb positive cells wereobserved following infection with the resulting stock. This indicatesthat the lacZ gene was packaged into an AAV virus which was incapable ofautonomous replication while residual adenovirus was completelyeliminated.

EXAMPLE 2 AAV Vectors Can Transfer and Stably Express A Foreign Gene inThe Adult Rat Brain

AAVlac was stereotaxically microinjected into various regions of theadult rat brain, including caudate nucleus, amygdala, striatum andhippocampus. Animals were initially sacrificed between 1 and 3 daysfollowing injection and sections were processed for X-galhistochemistry. Positive cells were demonstrated within each region. Theefficiency of gene transfer into the brain appeared to be at leastequivalent to that observed previously with HSV or adenovirus vectors.

In order to analyze the long-term stability of AAV gene transfer andexpression within the mammalian brain, animals were injected in thecaudate nucleus with AAVlac and sacrificed 2-3 months following surgery.First, the polymerase chain reaction (PCR) was adapted for use withinbrain sections to permit amplification and visualization of viral vectorDNA in situ. See Nuovo et al (1991) Am. J. Pathol. 139:1239-44; Nuovo etal (1993) PCR Meth, 2:305-12; Flotte et al (1993) Proc. Nat. Acad. Sci.USA 90:10613-17. Numerous cells within the brain were detected whichretained the bacterial lacZ gene after 2 months. There was no stainingon the opposite side of the brain sections or in sections processedwithout Taq polymerase, and positive cells were also absent from brainsinjected with adenovirus alone. Additional animals were then sacrificedand sections were processed for X-gal histochemistry (See Kaplitt et al(1991) Mol. Cell. Neurosci. 2:320-30) in order to identify cellscontaining functional β-galactosidase. Positive cells were identifiedwithin injected regions of the caudate nucleus up to 3 months followingvector injection. At no time were behavioral or physiologicalabnormalities detected within the animal subjects, and the brainsections showed no evidence of pathology resulting from the AAV genetransfer.

EXAMPLE 3 The AAV Vector Yields Expression of Tyrosine Hydroxylase inthe Caudate Nucleus of 6-OHDA Lesioned Rats

Parkinson's Disease (PD) is a neurodegenerative disorder characterizedby loss of the nigrostriatal pathway and is responsive to treatmentswhich facilitate dopaminergic transmission in the caudate-putamen. (Yahrand Bergmann, Parkinson's Disease (Raven Press, 1987), Yahr et al.(1969) Arch. Neurol. 21:343-54. In experimental animals, geneticallymodified cells that express tyrosine hydroxylase, and thereby synthesizediihydroxyphenylalanine (L-Dopa), induce behavioral recovery in rodentmodels of PD. (Wolff et al. (1989) PNAS (USA) 86:9011-14; Freed et al(1990) Arch. Neurol. 47:505-12: Jiao et al (1993) Nature 262:4505). Analternative approach is that of direct in vivo somatic cell genetransfer whereby the intrinsic cells of the neostriatum are convertedinto L-Dopa-producing cells by transduction with a vector expressing TH.An HSV-1 vector expressing TH has shown that this approach may be aviable alternative to tissue transplantation. (During et al. (1992) Soc.Neurosci Abstr. 18:331-8). However, HSV-1 vectors currently have severallimitations as described above. In order to generate a vector which mayhave therapeutic utility in human PD patients, we inserted a human THcDNA (form II) (O'Malley et al. Biochemistry 26:6910-14) into our AAVvector (AAVth). AAVth was packaged and helper virus was eliminated asdescribed above for AAVlac.

Unilateral 6-hydroxydopamine lesions of the substantia nigra have beenused to generate an established rodent model of PD. In this model, theasymmetry caused by differing postsynaptic receptor sensitivitiesbetween the denervated and intact striatum results in rotationalbehavior following systemic administration of dopaminergic agents, suchas apomorphine (Hefti et al. (1980) Brain Res. 195:123-7). The rate ofasymmetrical rotation is directly related to the severity of thestriatal dopamine deficit and this model has predictive ability indefining treatments which have therapeutic efficacy in PD. (Freed et al.(1987) Ann. Neurol. 8:510-19; Hargraves et al (1987) Life Sci.40:959-66). Lesioned rats were tested for apomorphine-induced rotationevery two weeks oil a minimum of three occasions, and animals thatsatisfied behavioral criteria of >95% lesion efficacy (>10rotations/minutes) were identified (Hefti et al. 1980). AAVth or AAVlacvirus, or vehicle alone (phosphate buffered saline, [PBS]), wasdelivered by stereotactic injection into the denervated striatum.Animals were tested for apomorphine-induced asymmetrical rotation at 2,4 and 9 weeks. The rotational behavior of the AAVlac injected animalswas similar to the PBS injected animals. In contrast, AAVth injectedanimals demonstrated significant behavioral recovery (FIG. 3), comparedto AAVlac or PBS injected groups (control groups). The averagebehavioral recovery caused by AAVth was 31±6% at 4 weeks and wasmaintained at 32±3% at 9 weeks (P<0.01) after injection.

In order to examine virally encoded TH gene expression followingtransduction with AAVth, animals were analyzed at times ranging from 24hours to 7 months after injection. Expression of TH from the AAV vectorwas detected using immunocytochemistry with a mouse monoclonal anti-THantibody. Although this antibody does not distinguish between the ratand human protein, TH is not expressed within either the intrinsicneurons or glia of the rat striatum (Chatterjee et al. (1992) Science258:1485-88). Furthermore, endogenous TH immunoreactivity (TH-IR) withinthe striatum is limited to the dopaminergic afferent fibers inunlesioned animals and is absent in the completely denervated striatum.In both control and AAVlac-injected rats there was no striatal THimmunoreactivity (TH-IR) on the denervated side. In contrast, in thedenervated striata injected with AAVth, numerous TH-IR cells wereclustered around the injection site and extending to 2 mm away from theinjection. The majority of cells within the striatum appeared to beneurons morphologically, and double-labelling with both the anti-THmonoclonal antibody and an anti-neurofilament antibody confirmed that asubstantial number of intrinsic striatal neurons expressedimmunoreactive TH de novo. Additional sections were then double labelledwith both the TH monoclonal antibody and a rabbit polyclonal antibody toglial fibrillary acidic protein (GFAP), a marker of astrocytes andoligodendrocytes. Other sections were double labelled with TH antibodyand antibodies for glutamic acid decarboxylase (GAD), a marker ofGABAergic neurons, the predominant neuronal population of theneostriatum. Double labelling revealed that the majority of TH-IR cellswere immunoreactive for GAD, while a small percentage of TH-IR cellswere GFAP positive. GABAergic neurons constitute approximately 95% ofthe intrinsic striatal neuron population with choline acetyl transferase(ChAT, cholinergic) positive cells making up the remainder. Doublelabelling with TH and ChAT also revealed expression of vector encoded THin striatal cholinergic neurons.

The titre of the AAVth stock used for these in vivo studies was 5×10⁶infectious particles (i.p.)/ml. Therefore a single injection of 2 μlwould result in 10,000 positive cells if the efficiency of infection was100% and each i.p. infected a different cell. However, as previousinfection of AAV does not prevent subsequent re-infection or multipleparticles infecting the same cell, in the immediate vicinity of theinjection we might expect cells to have multiple infection. Moreover,AAVth might also infect axons and terminals and following retrogradetransport be expressed in the cell body regions of the striatalafferents (e.g., the surviving dopamine nigral neurons, the cortex,reticular nucleus of the thalamus and dorsal raphe nuclei). In theAAVth-injected animals, the total number of striatal cells containingTH-IR consistently exceeded 1000 for each of the 2 μl injectionssuggesting a minimum of 10% in vivo efficiency, significantly greaterthan previous observations using defective HSV-1 vectors (@ 2%).Moreover, the level of expression was also examined at times rangingfrom 3 days to 7 months. Expression persisted throughout this 7 monthperiod, although the level of expression diminished by approximately50%.

Furthermore there were no signs of cytopathic effects. The only changesobserved in the short term animals (examined less than 1 week followinginjection) was a slight needle injury at the injection site which wassimilar in PBS-injected and AAVlac-injected animals. In the long-termanimals (greater than two months), the residual needle track was notconsistently visible and there was no evidence of any neuronal injury orreactive gliosis. There were also no behavioral or gross pathologicalsigns of brain damage in any subject.

EXAMPLE 4 Expression of Two Genes From a Single AAV Vector Results in DeNovo Synthesis of the Neurotransmitter Dopamine

Dopamine synthesis is catalyzed by two enzymes, TH and aromatic aciddecarboxylase (AADC). The reaction catalyzed by TH results in thesynthesis of L-Dopa, and this is the rate-limiting step in the synthesisof dopamine. Dopamine then results from conversion of L-Dopa by AADC.Although striatum does not contain cells which endogenously produce TH,there are a small percentage of striatal cells which produce AADC.Therefore, behavioral recovery in animals treated with AAVth (or otherapproaches using TH alone) presumably occurs secondary to conversion ofthe resulting L-Dopa to dopamine by endogenous striatal AADC. Since alimited number of cells produce AADC, however, it is possible thatsynthesis of dopamine could be enhanced by expression of both TH andAADC in every transduced cell. In this manner, any target cell wouldbecome an autonomous dopamine-producing cell following gene transfer.Recent evidence in fact suggests that expression of both genes in thedenervated striatum may be superior to expression of TH alone (Kang, et.al., 1993). Furthermore, the most substantial behavioral recoveryfollowing cell transplantation occurred when TH-expressing muscle cellswere utilized (Jiao et al 1993), and unlike fibroblasts from earlierstudies, muscle cells express endogenous AADC activity. This suggestedthat creation of an AAV vector containing both T14 and AADC would bevaluable.

Due to the limitation on insert sizes in AAV vectors, severalmodifications were required in order to create a vector containing bothgenes. First, the TH gene was truncated, eliminating the 5′ end.Truncation of the TH gene has actually been shown to increase enzymaticactivity due to removal of an amino terminal regulatory domain. (Walkeret al (1994) Bioch. Biophys. Acta 1206:113-119). Therefore, this serveda functional purpose as well as increasing the space available for othergenetic elements. In addition, a small synthetic oligonucleotide,encoding a novel epitope, was attached to the 5′ end of the truncatedTH. This novel epitope, termed “Flag”, is recognized by a commerciallyavailable monoclonal antibody; this provides an independent andunambiguous marker for expression of AAV-transduced T14 in vivo.

After modifying TH, the AADC gene was inserted into the vector. Creationof two independent expression units, with two promoters and twopolyadenylation signals, would have resulted in an insert size so largeas to be incompatible with the constraints of the AAV vector. Therefore,an IRES element was inserted between the Flag-TH and AADC cDNAs. Mosteukaryotic mRNAs are monocistronic; they contain a single-open readingframe, and when translation of a peptide is stopped and the ribosomefalls off of the transcript, additional downstream translational startsites cannot be utilized. When the IRES element is present on an mRNAdownstream of a translational stop codon, it directs ribosomal re-entry(Ghattas et al (1991) Mol. Cell. Biol. 11:5848-5959), which permitsinitiation of translation at the start of a second open reading frame(IRES=Internal Ribosome Entry Site). In this manner, a eukaryoticbicistronic mRNA can be created which allows translation of two distinctpeptides from a single mRNA (FIG. 2). Thus, with only a single promoter(CMV) and a single mRNA polyadenylation signal (SV40) directingexpression of a single transcript, translation of both the Flag-TH andAADC proteins could occur within a single cell transduced with a singleAAV vector.

Following creation of the plasmid AAV Flag-TH/AADC, each of theindependent expression parameters were tested in culture. The plasmidwas transfected into 293T cells, and then the following day thesubstrate tyrosine and an essential co-factor (tetrahydrobiopterin) wereadded to the tissue culture medium of some of these cultures. Forcomparison, additional cells were transfected with the plasmid AAVlac orwere mock-transfected. Samples of medium were obtained at 30 minutes and60 minutes after addition of co-factors (or mock treatment), and thesewere analyzed for the presence of dopamine by high-performance liquidchromatography (HPLC). As indicated in FIG. 6, very high levels ofdopamine were produced in 293T cells transfected with AAV Flag-TH/AADCin the presence of both co-factors. In similarly transfected cellslacking the co-factors, barely detectable amounts of dopamine wereproduced, while AAVlac-transfected or mock-transfected cells yieldedabsolutely no dopamine synthesis even in the presence of adequateco-factors. This indicated that 293T cells were incapable ofendogenously directing dopamine synthesis, however introduction of thebicistronic vector AAV Flag-TH/AADC converted these cells into highlevel, co-factor dependent producers of dopamine. Finally, it should benoted that these cells were then fixed and stained with the anti-Flagmonoclonal antibody, and this revealed highly specific histochemicaldetection of the Flag epitope with no background.

The specificity and function of the bicistronic AAV vector was furtheranalyzed in cultured 293T cells. Despite the above data, it was stillpossible that 293T cells contained endogenous AADC activity. If thiswere true, then expression of Flag-TH alone would have yielded similardata without achieving translation of the second (AADC) open-readingframe. In order to test this, an additional vector was created.AAVFlag-TH contains a monocistronic insert with the Flag-TH open readingframe but lacking both the IRES sequence and the AADC open readingframe. 293T cells were then transfected with AAV Flag-TH/AADC,AAVFlag-TH, AAVlac or no plasmid. Both co-factors were added to allcultures the following day, and then samples of the medium were testedfor both L-Dopa and dopamine by HPLC (Table 1). Cells which weretransfected with no plasmid or AAVlac could not synthesizeany-detectable level of either L-Dopa or dopamine. The lack of L-Dopademonstrated that 293T cells do not possess any endogenous TH activity.Furthermore, cells transfected with AAVFlag-TH yielded very high levelsof L-Dopa, but undetectable amounts of dopamine. This demonstrates that293T cells do not possess any AADC activity either. Furthermore, thisindicates that the truncated TH is highly active and the addition of the5′ Flag sequence did not adversely influence enzymatic activity.Finally, cells transfected with AAV Flag-TH/AADC produced significantamounts of L-Dopa but very high levels of dopamine. Presumably the lowerlevel of L-Dopa in these cells compared with those transfected withAAVFlag-TH was due to the efficient conversion of L-Dopa to dopamine.Thus two genes can be placed into a single AAV vector and techniquessuch as insertion of an intervening IRES sequence can result intranslation of both protein products. These data also indicate that AAVvectors can yield expression of multiple, functionally active proteinswhich can synergize in the production of a single, biologically activeneurotransmitter. The Flag epitope was also shown to be a specific,independent marker of AAV-derived TH protein production withoutadversely influencing TH enzymatic activity.

TABLE 1 Release of L-Dopa and Dopamine into the Culture Medium of 293TCells Following Plasmid Transfection L-Dopa (pg/ml) Dopamine (pg/ml)Blank <40 <40 LacZ <40 <40 Flag-TH 8200 <40 TH-AADC 800 4050

Controls which were transfected with pAAVlac or mock-transfected withPBS did not produce any detectable level of either L-Dopa or Dopamine,and therefore at the very least these cells did not contain TH activity.Following transfection with pAAV-FlagTH, which expresses only tyrosinehydroxylase, significant amounts of L-Dopa were produced but none wasconverted to dopamine. This demonstrated that the truncated TH with theN-terminal Flag epitope was enzymatically active, yet 293T cells containno endogenous AADC activity, hence the absence of conversion todopamine. By contrast, cells transfected with the bicistronic vectorpAAV-FlagTH-AADC yielded significant levels of L-Dopa, but far higherlevels of dopamine. This demonstrated that the Flag-TH was fully active,synthesizing L-Dopa, but that functional AADC was also translated fromthe same mRNA, and this converted much of the L-Dopa to dopamine.Therefore, two enzymes were expressed from a single vector, therebyconverting a cell with no endogenous TH or AADC activity into adopamine-producing cell.

EXAMPLE 5 AAV Vector-mediated Gene Therapy in a Primate Model ofParkinson's Disease

The great potential of the bicistronic vector as a therapeutic agent forParkinson's disease has led to the rapid initiation of primate studies.The primate model of Parkinson's disease is considered to be thegold-standard model for evaluation of potential therapies prior toentering human clinical trials. This model was originally developed fromthe observation in the early 1980s that groups of younger people weredeveloping a neurodegenerative disorder strikingly similar to idiopathicParkinson's disease. The source of this disorder was traced to the useof a street drug, and the specific causative agent was found to be1-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Langston (1985)Trends Pharmacol. Sci. 6:375-378). When MPTP was then given to primates,the animals developed a parkinsonian disorder that has become theprinciple model for testing anti-parkinsonian agents. Peripherallyadministered MPTP will cross the blood-brain barrier, whereupon it isconverted to MPP+ by monoamine oxidase B (MAO-B). This compound is thenselectively concentrated within the dopaminergic neurons of thesubstantia nigra via an energy-dependent, presynaptic uptake mechanism.This may be enhanced by the ability of neuromelanin, found within nigralneurons, to bind MPP+ (D'Amato et al (1986) Science 231:987-989). MPP+is a potent neurotoxin which eventually causes the degeneration ofnigral dopaminergic neurons and loss of the nigro-striatal dopaminepathway, as is seen in Parkinson's disease. (Redmond et al (1993) Ann.N.Y. Acad. Sci. 695:258-266; Tipton and Singer (1993) J. Neurochem.61:1191-1206).

Early studies initiated in MPTP primates have been designed to test thesafety of the AAV system in primates and to obtain information regardingthe potential therapeutic efficacy of AAV Flag-TH/AADC for Parkinson'sdisease. The initial study employed a small number of animals with onlymoderate nigral lesions and was designed to determine whether AAVvectors can transfer genes in the adult primate brain, and whetherdopamine transmission could be increased in the striatum using thebicistronic vector. Purified vector was stereotaxically injectedunilaterally into the striatum of MPTP-treated primates, and subjectswere then sacrificed either 10 days or 4.5 months after injection.Tissue sections were analyzed for Flag immunoreactivity, and numerouspositive cells were demonstrated in several sections from the injectedstriatum in both short and long-term subjects, while sections from theuninjected side were completely negative.

The majority of positive cells appeared morphologically to be neurons.This demonstrated for the first time that AAV vectors could successfullytransfer genes into the primate brain (During et al (1994) Abstr. Soc.Neurosci. 20:1465).

Biochemical analysis of tissue samples from treated primates furtherindicated that the vector did cause an increase in striatal dopamine(During et al (1994)). For example, in one subject sacrificed at 10 daysfollowing treatment, the level of dopamine from a striatal tissue samplenear the site of AAV injection was 18.93 ng/mg protein. An equivalenttissue sample from the uninjected, contralateral striatum yielded adopamine level of 7.97 ng/mg protein. Tissue samples from distal siteson the injected and uninjected sides resulted in dopamine levels of 2.48ng/mg and 2.27 ng/mg respectively. Since peripherally administered MPTPshould result in roughly equal lesions to the substantia nigrabilaterally, the approximately 140% increase in dopamine levels in theinjected striatum compared with the untreated side suggests that the AAVvector resulted in expression of functionally active enzymes.

A second study employed more severely lesioned primates in order todetermine whether there is a therapeutic potential for AAV Flag-TH/AADC.Subjects were divided into two groups, with the treated group receivingAAV Flag-TH/AADC and controls receiving AAVlac. All animals receivedbilateral stereotaxic injections, with the same virus infused into thestriatum on both sides of the brain. Subjects were then followed for 2.5months after surgery. Observations suggest that the bicistronic vectorresulted in sustained improvement in parkinsonian behavior (During et al(1994). Monthly assessments of control and treated animals by blindedcaretakers reported virtually no change in the behavior of animals whichwere subsequently determined to have been controls, while the responsein treated subjects varied from modest improvement to substantialrecovery of function. Most of the animals began the study unable,spending much of their time face-down and requiring assistance in orderto feed and groom themselves. Reports indicate that improvements intreated animals resulted in some cases in decreased time spent face-downand recovery of the ability to feed and groom themselves. These blindedobservations suggest that AAV vectors may result in behavioral recoveryof parkinsonian primates. It should also be noted that in both primatestudies, there was no behavioral or histological evidence of toxicitydue to the AAV vector. All of the data indicate that safe, long-termimprovement of human neurological diseases may be possible via geneticmodification of adult brain cells in vivo using AAV vectors.

EXAMPLE 6 Expression of a Growth Factor From an AAV Vector Can YieldRecovery of Function Following Neuronal Lesions

An additional AAV vector has been developed as an alternative approachto the treatment of Parkinson's disease. To date, the majority oftherapeutic strategies for PD have concentrated upon increasing striataldopamine levels. Although behavioral recovery in animal models has beenrepeatedly demonstrated, this is not a cure for the disease but rathersymptomatic palliation. Neuronal degeneration in the substantia nigra isthe pathological result of the disease process, and progression ofneurodegeneration is not altered by increasing striatal dopamine.Recently, however, several reports have determined that growth factorssuch as glial-derived neurotrophic factor (GDNF) can be protective ofand trophic for neurons of the substantia nigra (Lin et al (1993)Science 260:1130-1132). Therefore, an AAV vector was created containingthe cDNA for GDNF under the control of the CMV promoter.

Rats were lesioned with 6-OHDA and subsequently received injectionsAAVgdnf, AAVlac or saline into the lesioned substantia nigra (During etal (1994)). After several weeks, dopamine release into the striatum onthe lesioned side was determined using intracerebral microdialysis. Thistechnique permits sampling of local neurotransmitter release within aspecific brain region of living animals (During and Spencer (1993)Lancet 341:1607-1610). Baseline dopamine levels were sampled three timesand there was no difference between groups. Animals were then treatedwith potassium which induces release of dopamine from presynapticterminals. Neither the AAVlac nor saline treated animals showed anyvariation in dopamine release from baseline, indicating that there werefew dopaminergic terminals present within the striatum. The grouptreated with AAVgdnf, however, yielded an significant increase indopamine release of 200% (p<0.05). Since the AAV vector only containedthe gene for a growth factor, the restoration of potassium-induceddopamine release into the striatum suggests that GDNF expression eitherprotected or promoted regrowth of dopaminergic neurons in the substantianigra following 6-OHDA treatment.

These results were further supported by subsequent administration ofnomifensine to animal subjects after dopamine levels in the AAVgdnfgroup returned to baseline. Nomifensine is a drug which increasessynaptic dopamine levels by inhibiting dopamine-reuptake. Again bothcontrol groups showed no change in dopamine levels in response tonomifensin, while striatal dopamine increased 150% (p<0.05) in the grouptreated with AAVgdnf. Together these data demonstrate that AAV-mediatedtransfer of a growth factor gene can either protect or restoredopaminergic inputs to the striatum. Thus gene therapy can be useful forboth palliation of PD through striatal expression of synthetic enzymesfor dopamine as well as for treatment of the underlying disease processby expressing growth factors which may protect or regeneratedopaminergic neurons. The present invention is the first demonstrationthat AAV vectors can safely and efficiently transfer and express aforeign gene marker gene (lacZ) in the adult rat brain. Furthermore,stability of viral DNA and lacZ expression within the brain was observedfor at least 7 months with no evidence of pathology or toxicity.Expression of human tyrosine hydroxylase (hTH) was demonstrated in bothneurons and glia of rat brains which had previously received unilateral6-hydroxydopamine (6-OHDA) lesions in the substantia nigra. Theselesions result in asymmetrical (contralateral to the side of the lesion)rotational behavior when rats are treated with apomorphine, and this hasbeen used as a behavioral model of Parkinson's disease (PD). Followingvector injection into the caudate nucleus, expression of hTH wasdemonstrated up to 7 months later, and preliminary evidence indicatesthat sustained expression of hTH from an AAV vector can reducerotational behavior following 6-OHDA administration.

2 22 base pairs nucleic acid single linear DNA (genomic) YES NO unknown1 CCGACTGATG CCTTCTGAAC AA 22 22 base pairs nucleic acid single linearDNA (genomic) YES YES unknown 2 GACGACAGTA TCGGCCTCAG GA 22

What is claimed is:
 1. A method for ameliorating a symptom of a centralnervous system disorder in a mammal, the method comprising, directadministration of an adeno-associated virus-derived vector to a targetcell in the brain of the mammal, said vector comprising a DNA sequence,wherein said DNA sequence is exogenous to an adeno-associated virus andcomprises a sequence encoding a therapeutic protein in operable linkagewith a promoter sequence, wherein said adeno-associated virus-derivedvector is free of both wild-type and helper virus, and wherein theexogenous DNA sequence is expressed in the target cell for greater than3 months such that the symptom of the central nervous system disorder isameliorated.
 2. The method of claim 1, wherein the exogenous DNAsequence is expressed in said target cell either constitutively or underregulatable conditions.
 3. The method of claim 2, wherein the exogenousDNA sequence encodes a protein selected from the group consisting oftyrosine hydroxylase and aromatic amino acid decarboxylase.
 4. Themethod of claim 2, wherein the exogenous DNA sequence encodes a specificneurotrophic factor for mesencephalic dopaminergic neurons.
 5. Themethod of claim 4, wherein the specific neurotrophic factor formesencephalic dopaminergic neurons is selected from the group consistingof glial-derived neurotrophic factor (GDNF), brain-derived neurotrophicfactor (BDNF), and nerve growth factor (NGF).
 6. The method of claim 1,wherein all adeno-associated viral genes of the vector have been deletedor inactivated.
 7. The method of claim 1, wherein the vector comprisesonly the inverted terminal repeats of adeno-associated virus.
 8. Themethod of claim 1, wherein said promoter sequence is a central nervoussystem-specific promoter.
 9. The method of claim 8, wherein saidpromoter sequence is a neuron-specific promoter.
 10. The method of claim8, wherein said promoter sequence is a glia-specific promoter.
 11. Themethod of claim 1, wherein the target cell is in a region of the brainselected from the group consisting of cerebellum, striatum, hippocampus,and substantia nigra.
 12. The method of claim 1, wherein the target cellis a primate target cell or a rat target cell.
 13. The method of claim12, wherein the primate target cell is a human target cell.
 14. Themethod of claim 1, wherein direct administration is by stereotaxicinjection.
 15. An adeno-associated virus-derived vector comprising onlythe replication and packaging signals of adeno-associated virus, andfurther comprising a DNA molecule comprising a DNA sequence encodingtyrosine hydroxylase, a DNA sequence encoding aromatic amino aciddecarboxylase, and a promoter sequence.